VEGF-C在淋巴管生成及乳腺癌淋巴道转移中的作用

目的： 探讨阻断VEGF-C/Flt-4调控系统对淋巴管生成和乳腺癌淋巴道转移的影响。方法： 体外培养胎牛胸导管内皮细胞，观察VEGF-C和抗Flt-4抗体对淋巴管内皮细胞增殖的影响；设计合成VEGF-C反义脱氧寡核苷酸（ASODN），体外实验观察其对VEGF-C基因表达的影响；建立乳腺癌裸鼠原位移植瘤模型并观察ASODN对肿瘤淋巴管生成及肿瘤生长转移的影响。结果： 加入前列腺癌细胞PC3(高表达VEGF-C)上清后，淋巴管内皮细胞增殖活跃；加入抗Flt-4抗体后各时段细胞计数均明显少于其它各组。体外实验RT-PCR及 Western blotting显示ASODN作用的MCF-7细胞组VEGF-C mRNA及蛋白表达均低于对照组。体内RT-PCR检测表明ASODN组移植瘤VEGF-C mRNA表达明显受抑制；5-Nase-ALPase双重酶组织化学法结果显示ASODN组移植瘤的淋巴管生成明显减少；ASODN组肿瘤生长速度较对照组缓慢，且肿瘤体积、淋巴结转移明显低于对照组。结论： VEGF-C/Flt-4调控系统与乳腺癌组织的淋巴管生成及肿瘤的淋巴道转移密切相关。阻断淋巴管内皮细胞Flt-4表达，可在一定程度上抑制肿瘤细胞诱导的淋巴管内皮细胞增殖；ASODN通过下调乳腺癌VEGF-C的表达，减少肿瘤淋巴管的生成及淋巴结转移。     

人参皂甙Rh2对小鼠移植瘤血管内皮生长因子C及淋巴管生成的影响

目的 观察人参皂甙Rh2对小鼠移植瘤生长,肿瘤细胞血管内皮生长因子c(VEGF-C)表达和淋巴管密度的影响,探讨其作用机制.方法 用S180瘤株构建55只小鼠移植瘤模型,成瘤后灌服人参皂甙Rh2,观察用药组与对照组移植瘤的生长情况,免疫组织化学染色,比较用药2周、3周后癌细胞VEGF-C的表达及LYVE-1标记的淋巴管密度与对照组的差异.结果接种约第3周开始,对照组移植瘤生长速度明显快于用药组.用药第2周癌细胞VEGF-C表达及淋巴管密度与对照组无差异;第3周VEGF-C表达较对照组弱,淋巴管密度也较对照组低,有差异(P<0.05). 结论 人参皂甙Rh2能抑制肿瘤生长,降低淋巴管密度,其机制可能是通过降低VEGF-C在癌细胞的表达,干扰淋巴管的生成.     

大肠癌组织VEGF-C、COX-2、Flt-4及nm23表达与淋巴管生成及淋巴结转移的关系

目的:探讨结直肠癌组织中血管内皮生长因子-C(VEGF-C)、环氧化酶-2(COX-2)、酪氨酸激酶受体(Flt-4)及其转移抑制基因nm23的表达与淋巴管生成、淋巴结转移的关系。方法:选取2005-09/2009-09南昌大学第二附属医院病理科大肠癌手术切除标本94例,采用酶组织化学方法及SP免疫组化法对大肠癌组织中VEGF-C、COX-2、Flt-4和nm23的表达及淋巴管的生成进行观察,分析大肠癌组织中VEGF-C、COX-2、Flt-4及nm23的表达与淋巴管生成及淋巴结转移之间的关系。结果:VEGF-C、COX-2、Flt-4及nm23在大肠癌和癌旁正常肠黏膜组织中的表达比较均具有统计学差异(P0.05)。VEGF-C及Flt-4的表达与Dukes分期、淋巴管计数和淋巴结的转移有显著相关性(P0.05);COX-2的表达与肿瘤大小、分化程度、浸润深度、肿瘤的分期、淋巴管计数和淋巴结的转移有显著相关性(P0.05);nm23的表达与分化程度、Dukes分期和淋巴结的转移有显著相关性(P0.05)。结论:VEGF-C、COX-2及Flt-4的表达与大肠癌淋巴管生成和淋巴结转移密切相关;nm23基因在大肠癌的浸润和淋巴结转移过程中起负性调控作用。     

D-Limonene对小鼠移植瘤生长及淋巴管生成的影响

目的:观察右旋柠烯对小鼠移植瘤生长及淋巴管生成的影响,并探讨其作用机制。方法:皮下注射肉瘤(S180)腹水型瘤株构建小鼠移植瘤模型,给予D-Lim onene干预,免疫组化染色观察瘤细胞V EG F-C表达,LY V E-1标记淋巴管,观察其分布。结果:对照组瘤细胞V EG F-C表达较强,瘤周边部淋巴管较多,有淋巴结、肺转移。用药组瘤细胞V EG F-C表达较弱;瘤周边部淋巴管较少,未见淋巴结、肺转移。结论:D-Lim onene有抑制移植瘤内瘤细胞V EG F-C表达和淋巴管生成的作用,有可能降低肿瘤的淋巴道转移。     

表没食子儿茶素-3-没食子酸酯抑制乳腺癌裸鼠移植瘤淋巴管生成

目的 探讨表没食子儿茶素-3-没食子酸酯(EGCG)对人乳腺癌裸鼠皮下移植瘤淋巴管生成的影响及其作用机制.方法 建立裸鼠皮下移植瘤模型30例,随机分成5组,即生理盐水组、5-氟脲嘧啶(5-FU)组、20mg/kg EGCG组、10mg/kg EGCG组、5mg/kg EGCG组,观察瘤组织血管内皮生长因子C(VEGF-C)的表达情况,淋巴管内皮细胞透明质酸受体1(LYVE-1)标记淋巴管,检测淋巴管密度及面积;Western blotting检测移植瘤组织VEGF-C蛋白的表达情况.结果 免疫组织化学染色VEGF-C在20mg/kg EGCG处理组中的表达量明显低于生理盐水组和5-FU组,且VEGF-C的表达与EGCG成剂量依赖性;移植瘤周边淋巴管密度、面积在20mg/kg EGCG处理组中显著低于5-FU组和生理盐水组,差异有统计学意义;Western blotting结果显示,EGCG高剂量处理组VEGF-C蛋白明显低于生理盐水组.结论 EGCG可以抑制乳腺癌裸鼠移植瘤中VEGF-C的表达及淋巴管的生成.     

Smad4的表达与胰腺癌淋巴管生成之间的关系

目的观察血管内皮生长因子(VEGF)-C和Smad 4在胰腺癌组织内的表达情况,分析VEGF-C和Smad 4的表达与胰腺癌淋巴管生成及淋巴结转移之间的关系。方法取胰腺癌病例52例,其中,无淋巴结转移组12例,淋巴结转移组40例。应用免疫组化和Western blot技术检测VEGF-C和Smad4在胰腺癌组织内的表达情况。以D2-40作为淋巴管内皮标记物,观察胰腺癌组织内淋巴管生成情况。结果 VEGF-C主要表达于胰腺癌细胞胞质内,在淋巴结转移组的表达水平明显高于无淋巴结转移组。Smad4表达于胰腺癌细胞胞质和胞核内,在无淋巴结转移组的表达水平明显高于淋巴结转移组,Smad4表达阳性组淋巴管数密度明显低于Smad4表达阴性组(=0.02)。Smad4的表达与VEGF-C的表达呈显著的负相关性。结论 VEGF-C在胰腺癌淋巴管的发生及淋巴结转移中发挥重要作用,Smad4可能通过调节VEGF-C蛋白的表达抑制胰腺癌淋巴管生成和淋巴结转移。     

COX-2抑制剂对肺癌裸鼠移植瘤血管生成素基因表达的影响

目的：探讨环氧化酶-2(COX-2)抑制剂尼米舒利（NIM）对肺癌裸鼠移植瘤的血管生成素基因表达的影响及意义。 方法： 人肺癌A549细胞接种于裸鼠皮下，建立肺癌裸鼠移植瘤模型并予NIM治疗，计算NIM的抑瘤率，RT-PCR检测裸鼠移植瘤组织血管生成素-1、2 (Ang-1、Ang-2) mRNA表达, 免疫组织化学法测定裸鼠移植瘤组织微血管密度(MVD)。 结果： NIM可有效抑制裸鼠移植瘤的生长，其抑瘤率为43.02%。 NIM治疗组裸鼠移植瘤组织Ang-2 mRNA水平显著低于对照组（P<0.01），Ang-1 mRNA水平无显著改变（P>0.05）, Ang-2/Ang-1 mRNA比值下降（P<0.01）；同时MVD明显低于对照组（P<0.01）。 结论： COX-2抑制剂NIM可下调Ang-2基因表达,改变Ang-2/Ang-1 mRNA比值，该作用可能是COX-2抑制剂抑制肿瘤血管生成从而抑制肿瘤生长的机制之一。     

选择性环氧合酶-2抑制剂Celebrex对胰腺癌新生血管生成的影响

探讨选择性环氧合酶-2(COX-2)抑制剂Celebrex对裸鼠胰腺癌PC-3细胞移植瘤生长和肿瘤组织新生血管生成的影响,为Celebrex的临床应用提供依据.本文采用体外观测Celebrex对移植瘤生长的影响,并选用Ⅳ型胶原作为肿瘤微血管标记物,测定移植瘤中微血管密度(MVD).结果显示,治疗组移植瘤生长曲线较对照组明显低平,对照组移植瘤近似体积为0.438 cm3,治疗组为0.212 cm3(抑瘤率为51.6%,P＜0.05).对照组肿瘤组织MVD为63.87±13.67,治疗组32.25±12.99,两组间差异有非常显著性(P＜0.01).表明COX-2可能参与了肿瘤新生血管的生成,选择性COX-2抑制剂Celebrex则具有抑制肿瘤生长和对抗肿瘤新生血管生成的作用.     

Smad4和VEGF-C在喉癌中的表达及其与淋巴管生成之间的关系

目的观察Smad4与VEGF-C在喉癌组织内的表达情况,分析Smad4和VEGF-C的表达与喉癌组织内淋巴管生成及淋巴结转移之间的关系。方法取喉癌病例58例,其中淋巴结转移组34例,无淋巴结转移组24例。应用免疫组化法和Western blot技术观察Smad4和VEGF-C在喉癌组织内的表达。以D2-40特异性标检测喉癌组织内淋巴管生成情况。结果 Smad4在无淋巴结转移的喉癌组织内的表达率明显高于其在有淋巴结转移组的表达率。Smad4表达阳性组的淋巴管数密度(LVD)明显低于Smad4表达阴性组的LVD。VEGF-C在淋巴结转移组的表达率明显高于其在无淋巴结转移组的表达率。Smad4的表达与VEGF-C的表达呈显著的负相关性(r=-0.391)。结论 VEGF-C在喉癌淋巴管的发生及淋巴结转移中发挥重要作用。Smad4与VEGF-C的表达呈负相关,Smad4可能有抑制喉癌淋巴管生成和淋巴道转移的作用。     

VEGF-C表达与胰腺癌淋巴结转移及预后的关系

目的 观察血管内皮生长因子(VEGF)-C在胰腺癌组织内的表达情况,分析VEGF-C的表达与胰腺癌淋巴结转移和预后之间的关系。方法 取胰腺癌病例52例,其中,伴淋巴结转移组36例,无淋巴结转移组16例。应用免疫组化法和Western blot技术观察VEGF-C在胰腺癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,观察胰腺癌组织内淋巴管生成的情况。采用Kaplan-Meier法绘制生存曲线判断VEGF-C的表达对胰腺癌预后的影响。结果 Western blot和免疫组化法检测结果表明,VEGF-C主要表达于胰腺癌细胞浆内,淋巴结转移组阳性表达量明显高于无淋巴结转移组(p<0.05)。D2-40表达于胰腺癌组织内淋巴管内皮细胞,VEGF-C阳性组淋巴管数密度明显高于VEGF-C阴性组(p<0.05),表明VEGF-C的表达与胰腺癌淋巴管生成密切相关。Kaplan-Meier生存分析表明VEGF-C表达阴性患者的生存率均高于VEGF-C表达阳性患者,VEGF-C的表达影响患者的预后。结论 VEGF-C在胰腺癌的淋巴管生成和淋巴结转移过程中发挥重要作用,VEGF-C的表达是影响胰腺癌患者预后的主要因素之一。     

Effect of celecoxib combined with chemotherapy drug on malignant biological behaviors of gastric cancer

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been reported to have antitumor effects. In some tumor models, the combination of celecoxib with chemotherapy agents has shown synergistic antitumor effect; however, the effect of celecoxib combination with tegafur/gimeracil/oteracil potassium on the malignant biological behaviors of gastric cancer in nude mice is unclear. In this study, female nude mice were subcutaneously transplanted with SGC-7901 gastric cancer cells. When the tumor model formed, the mice were divided into control group, celecoxib group, tegafur/gimeracil/oteracil potassium group, and the combination of both drug regimens group. Mice were treated for 3 weeks. Following treatment, the proliferating index was calculated, apoptosis related proteins, COX-2, vascular endothelial growth factor-C (VEGF-C) and lymphatic vessel density were quantified in tumor tissues by immunohistochemistry. Apoptosis was evaluated by TUNEL staining. The results revealed that celecoxib and tegafur/gimeracil/oteracil potassium alone significantly inhibited tumor growth. The combination of these two drugs showed a synergistic antitumor effect. Both celecoxib and tegafur/gimeracil/oteracil potassium alone inhibited proliferation and promoted apoptosis. The combination of these two drugs further enhanced this anticancer effect. Both celecoxib and the combination treatment inhibited lymphangiogenesis and the expression of COX-2 and VEGF-C. However, tegafur/gimeracil/oteracil potassium treatment had no obvious effect on lymphangiogenesis. These results suggested that the combination of celecoxib and tegafur/gimeracil/oteracil potassium produced a synergistic antitumor effect, possibly by inhibiting the proliferation of tumor cells and promoting apoptosis. Celecoxib and celecoxib in combination with tegafur/gimeracil/oteracil potassium possibly by reducing the expression of COX-2, in turn down-regulating the expression of VEGF-C, resulted in the inhibition of lymphangiogenesis.     

Role of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary lymphedema

The pathophysiology of secondary lymphedema remains poorly understood. To clarify the roles of cyclooxygenase (COX)-2 in enhancement of lymphangiogenesis during secondary lymphedema, we tested a mouse tail model and evaluated the recurrence of lymph flow. To induce lymphedema, a circumferential incision was made in the tail of anesthetized mice to sever the dermal lymphatic vessels. The maximum diameters of the tails were measured weekly. We found that the diameters of the tails around the wounds were markedly increased after surgery, and reached maximum size 2 weeks after wounding in mice without a COX-2 inhibitor, celecoxib (Celecoxib-). Expression of COX-2 in wound granulation tissues was markedly increased 1 week after surgery compared with unwounded naive control mice. In Celecoxib-, recurrence of lymphatic flow in the wound granulation tissues was detected 3 weeks after surgical treatment. In contrast, lymphatic flow was markedly suppressed in mice treated with celecoxib (Celecoxib+). Newly formed lymphatic structures were identified in the granulation tissues formed at wounded lesions in Celecoxib-, whereas those were markedly suppressed in Celecoxib+. Interstitial tissue pressures in the distal areas of the tail wounds were markedly increased in Celecoxib+ with reduced expression of vascular endothelial cell growth factor (VEGF)-C. F4/80-positive cells were accumulated to the wound granulation tissues in Celecoxib-, and the accumulation of these cells was suppressed in Celecoxib+. Prostaglandin E(2) (PGE(2)) upregulated the expressions of VEGF-A and VEGF-C in cultured macrophages, but not human lymphatic microvascular endothelial cells. The present study therefore suggests that lymphangiogenesis, together with recurrence of lymph flow after surgical induction of lymphedema, is upregulated by COX-2 possibly via generation of PGs.     

Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.     

VEGF-C在卵巢癌局部淋巴结内淋巴管生成中的作用

目的观察血管内皮生长因子-C(VEGF-C)在卵巢癌组织内的表达,分析其与卵巢癌局部淋巴结内淋巴管生成之间的关系。方法取卵巢癌64例,其中,有淋巴结转移40例,无淋巴结转移24例。应用免疫组化法和Western blot技术观察VEGF-C在卵巢癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,检测卵巢癌局部淋巴结内淋巴管生成情况。结果 VEGF-C主要表达于卵巢癌细胞浆和胞膜以及癌组织周围浸润的炎性细胞,在有淋巴结转移组的表达率明显高于其在无淋巴结转移组的表达率。Western blot检测结果表明,VEGF-C蛋白在有淋巴结转移的卵巢癌组织中的表达量高于其在无淋巴结转移的卵巢癌组织内的表达量。D2-40表达于卵巢癌局部淋巴结内的淋巴管内皮细胞,在有转移的淋巴结内可见大量新生的淋巴管,淋巴管腔内存在入侵的肿瘤细胞,在无转移的淋巴结内观察到新生的淋巴管。在无淋巴结转移组病例中,卵巢癌组织VEGF-C阳性者局部淋巴结内淋巴管密度明显高于VEGF-C阴性者淋巴结内的淋巴管密度。结论 VEGF-C的表达与卵巢癌淋巴结转移密切相关,卵巢癌在发生局部淋巴结转移之前存在淋巴结内淋巴管生成的现象,卵巢癌组织内VEGF-C的表达在卵巢癌局部淋巴结内的淋巴管生成中可能发挥重要作用。     

Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model

We reported that cyclo-oxygenase (COX)-2 expression in human breast cancer stimulated cancer cell migration and invasiveness, production of vascular endothelial growth factor (VEGF)-C and lymphangiogenesis in situ, largely from endogenous PGE2-mediated stimulation of prostaglandin E (EP)1 and EP4 receptors, presenting them as candidate therapeutic targets against lymphatic metastasis. As human breast cancer xenografts in immuno-compromised mice have limitations for preclinical testing, we developed a syngeneic murine breast cancer model of spontaneous lymphatic metastasis mimicking human and applied it for mechanistic and therapeutic studies. We tested the roles of COX-2 and EP receptors in VEGF-C and -D production by a highly metastatic COX-2 expressing murine breast cancer cell line C3L5. These cells expressed all EP receptors and produced VEGF-C and -D, both inhibited with COX-2 inhibitors or EP4 (but not EP1, EP2 or EP3) antagonists. C3H/HeJ mice, when implanted SC in both inguinal regions with C3L5 cells suspended in growth factor-reduced Matrigel, exhibited rapid tumor growth, tumor-associated angiogenesis and lymphangiogenesis (respectively measured with CD31 and LYVE-1 immunostaining), metastasis to the inguinal and axillary lymph nodes and the lungs. Chronic oral administration of COX-1/COX-2 inhibitor indomethacin, COX-2 inhibitor celecoxib and an EP4 antagonist ONO-AE3-208, but not an EP1 antagonist ONO-8713 at nontoxic doses markedly reduced tumor growth, lymphangiogenesis, angiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in responding mice revealed reduced VEGF-C and -D proteins, AkT phosphorylation and increased apoptotic/proliferative cell ratios consistent with blockade of EP4 signaling. We suggest that EP4 antagonists deserve clinical testing for chemo-intervention of lymphatic metastasis in human breast cancer.     

Smad4的表达与卵巢癌淋巴管生成及淋巴结转移之间的关系

目的观察Smad4和血管内皮生长因子(VEGF)-C在卵巢癌组织内的表达情况,分析Smad4和VEGF-C的表达与卵巢癌淋巴管生成及淋巴结转移之间的关系。方法取卵巢癌病例60例,其中,淋巴结转移组36例,无淋巴结转移组24例。应用免疫组化法和Westernblot技术观察Smad4和VEGF-C在卵巢癌组织内的表达。以D2-40作为淋巴管内皮特异性标记物,检测卵巢癌组织内淋巴管生成情况。结果 Smad4表达于卵巢癌细胞胞浆和胞核内,其在无淋巴结转移组的表达率明显高于其在有淋巴结转移组的表达率。Smad4表达阳性组的淋巴管数密度(LVD)明显低于Smad4表达阴性组的LVD。VEGF-C主要表达于卵巢癌细胞胞浆内,其在淋巴结转移组的表达率明显高于其在无淋巴结转移组的表达率。Smad4的表达与VEGF-C的表达呈显著的负相关性。Western blot检测结果表明,VEGF-C蛋白在有淋巴结转移卵巢癌组织中的表达量高于其在无淋巴结转移卵巢癌组织内的表达量,而Smad4在有淋巴结转移卵巢癌组织内的表达量明显低于其在无淋巴结转移组的表达量。结论 Smad4与VEGF-C的表达呈负相关,Smad4可能通过调节VEGF-C蛋白的表达而抑制卵巢癌淋巴管生成和淋巴道转移。